Many genetic diseases and cancers are caused by inappropriate transcription of certain genes, which can be affected by DNA topology. DNA topoisomerases that regulate the cellular DNA topology in vivo are the target of many anti-cancer drugs. It has been recognized that transcription and DNA supercoiling are mutually linked. Therefore, a detailed mechanistic study of transcription-coupled DNA supercoiling and its biological functions is needed. The long-term goal of the proposed research is to understand how transcription affects DNA topology, chromosome structure, and the coupled DNA transactions. Specific aims are: (1) In vitro biochemical studies to directly test the "twin-supercoiled-domain" model of transcription. Different aspects of transcription-coupled DNA supercoiling, especially the stimulation by sequence-specific DNA-binding proteins, will be explored. Results are expected to provide a basis to propose a detailed mechanism to explain transcription-coupled DNA supercoiling. (2) Studies of transcription-coupled DNA supercoiling in E. coil cells. A two plasmid system will be designed to study transcription-coupled DNA supercoiling in vivo. These studies will mirror the in vitro studies for supporting the proposed mechanism. (3). Design and synthesis of novel DNA templates to study DNA supercoiling and transcription-coupled DNA supercoiling. In the proposed studies, two types of novel DNA templates will be designed and synthesized. The first type of DNA templates are circular DNA molecules with one or two interstrand covalent cross-links. The second type of DNA templates are linear supercoiled DNA molecules. These proposed novel supercoiled DNA templates will provide new tools to study DNA supercoiling and transcription-coupled DNA supercoiling. Results of these combined studies are anticipated to significantly enhance our understanding of the mechanism of transcription-coupled DNA supercoiling and its roles in other DNA transactions such as DNA replication. Models will be proposed to explain transcription-coupled DNA supercoiling both in vitro and in vivo.